JPWO2004009639A1 - Single chain antibody and its use - Google Patents

Abstract

The present invention provides a single-chain antibody that retains its specific binding activity with an antigen, and a labeled single-chain antibody in which a labeling substance is bound to the single-chain antibody. Specifically, the labeled single chain antibody of the present invention can be produced by binding a labeling substance to the linker portion of the single chain antibody. The antibody is produced using a cell-free protein synthesis system derived from wheat germ, and the antibody is produced in a low reducing state in which the intramolecular disulfide bond is retained. Further, a method for producing a solid-phase immobilized single chain antibody by binding the antibody to a solid phase via a labeling substance and a method for analyzing an antigen-antibody reaction using the solid-phase immobilized single chain antibody are performed.

Description

  This application claims the priority from Japanese Patent Application No. 2002-210067, which is incorporated herein by reference.

  The present invention relates to a single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker, and a labeled single-chain antibody characterized by having a labeling substance in the linker portion of the antibody, and their Regarding usage etc.

The single-chain antibody is characterized by being able to reduce non-specific binding to cells because it has a small size consisting of only the antigen-binding region as compared with complete IgG. When a single-chain antibody is used for analysis of an antigen-antibody reaction, various labeling methods have been developed for the antibody in order to trace the immune reaction (Cloutier, SM et al., Mol. Immunol., 37, 1067-1077 (2000)). As a method of labeling an antibody, a method of binding biotin or the like to the C-terminus or N-terminus of the antibody with biotin ligase (Cloutier, SM, et al., Mol. Immunol., 37, 1067-1077 (2000)) ) And the like have been proposed, but there has been a problem in that the labeling reduces the binding activity of the antibody with the antigen.
Further, in recent years, the development of a technique for immobilizing such an antibody on a chip, a bead or the like for the purpose of detecting a large amount of a specific antigen existing on the cell surface rapidly (Mitchell, P., Nature) has been remarkable. Biotechnology, 20, 225-229 (2002)). Specifically, the microspotting method, the microprinting method, the chemical modification method and the like are used, but all of them have problems in that the binding activity of an antibody to an antigen is decreased, and it is difficult to achieve high density. there were.
On the other hand, a method has also been proposed in which a substance having a specific binding property such as streptavidin/biotin that is covalently bonded to a solid phase substrate of a protein is bonded as a linker. However, even in this method, there is no example of immobilizing the antibody while retaining the binding property to the antigen.

The present invention is a single-chain antibody having a structure in which the heavy chain and the light chain of the antibody are cross-linked through a linker, which retains the activity of binding to an antigen, and a labeled single-chain antibody obtained by labeling the antibody and The purpose is to provide a method of utilizing. Further, a method for immobilizing the antibody while retaining the binding property of the antibody to the antigen, a labeled single-chain antibody for use in the method, and a method for analyzing an antigen-antibody reaction using the labeled single-chain antibody The purpose is to provide.
The present inventors have conducted extensive studies in order to solve the above-mentioned problems, and as a result, bind biotin to the linker portion of a single-chain antibody in which the heavy chain and the light chain of the antibody are bound via a linker to give streptavidin. The single-chain antibody was brought into contact with the substrate coated on the surface, and the antibody was bound to the substrate. When an antigen was brought into contact with the solid-phase immobilized single-chain antibody thus produced, it was found that the binding property of the antibody to the antigen was extremely high. The present invention has been accomplished based on these findings.
That is, the present invention is
1. A single-chain antibody characterized by having a structure in which a heavy chain and a light chain of an antibody are cross-linked through a linker, or a labeled single-chain antibody characterized by carrying a labeling substance on the linker portion of the single-chain antibody .
2. A single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are crosslinked via a linker, or a labeled single-chain antibody having a labeling substance carried on the linker portion of the single-chain antibody, wherein the heavy chain and the light chain of the antibody Is a variable region. A single-chain antibody or a labeled single-chain antibody.
3. A labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker and carrying a labeling substance on the linker part, wherein the labeling substance is an antibody in the presence of a specific enzyme. A labeled single-chain antibody, which is a substance capable of binding to the polypeptide of the linker moiety of.
4. In a labeled single-chain antibody having a structure in which a heavy chain and a light chain that are variable regions of an antibody are cross-linked via a linker, and a labeling substance is carried on the linker portion, the labeling substance is a specific enzyme. A labeled single-chain antibody, which is a substance capable of binding to a polypeptide of a linker portion of an antibody in the presence thereof.
5. In a labeled single chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker, and carrying a labeling substance on the linker part, the labeling substance is a part of the linker part of the antibody. A labeled single-chain antibody characterized by being incorporated.
6. In a labeled single-chain antibody having a structure in which a heavy chain and a light chain, which are variable regions of an antibody, are cross-linked via a linker, and the linker moiety carries a labeling substance, the labeling substance is a linker part of the antibody. A labeled single chain antibody characterized in that it is incorporated as a part of
7. Labeling having a structure in which the heavy chain and the light chain of an antibody are crosslinked via a linker, and carrying a labeling substance capable of binding to the polypeptide of the linker part of the antibody in the linker part in the presence of a specific enzyme A single-chain antibody, wherein the labeling substance is biotin and the enzyme is biotin ligase.
8. The variable region of the antibody has a structure in which heavy and light chains are cross-linked via a linker, and a labeling substance capable of binding to the polypeptide of the linker part of the antibody in the presence of a specific enzyme is provided in the linker part. The labeled single-chain antibody carried, wherein the labeling substance is biotin and the enzyme is biotin ligase.
9. The single chain antibody or the labeled single chain antibody according to any one of the above items 1 to 8, which has a Kd value equivalent to that of a natural antibody and is produced by a cell-free protein translation system using wheat germ.
10. A DNA characterized in that DNAs encoding heavy and light chains of an antibody having a binding property to a specific antigen are linked via a DNA encoding a linker.
11. In the DNA in which the DNAs encoding the heavy chain and the light chain of the antibody having the ability to bind to a specific antigen are linked via the DNA encoding the linker, the heavy chain and the light chain of the antibody are variable regions DNA characterized by:
12. In the DNA in which the DNAs encoding the heavy chain and the light chain of the antibody having the binding property to the specific antigen are linked via the DNA encoding the linker, the DNA encoding the linker is A DNA comprising a base sequence capable of binding a labeling substance in the presence thereof.
13. In a DNA in which heavy chain and light chain that are variable regions of an antibody capable of binding to a specific antigen are linked via a DNA encoding a linker, the DNA encoding the linker is post-translationally translated. A DNA comprising a base sequence capable of binding a labeling substance in the presence of a specific enzyme.
14. DNAs encoding heavy and light chains of an antibody having a binding property to a specific antigen are linked via a DNA encoding a linker containing a base sequence capable of binding a labeling substance after translation in the presence of a specific enzyme. DNA having a nucleotide sequence capable of binding the labeling substance encodes an amino acid sequence recognized by biotin ligase.
15. DNA encoding a heavy chain and a light chain, which are variable regions of an antibody capable of binding to a specific antigen, and a linker containing a base sequence capable of binding a labeling substance in the presence of a specific enzyme after translation. A DNA characterized in that, in the DNA linked via, the base sequence capable of binding the labeling substance encodes an amino acid sequence recognized by biotin ligase.
16. A method for producing a labeled single chain antibody, which comprises transcribing and translating the DNA according to any one of the above items 10 to 15 using a protein synthesis system in the presence of a labeling substance and a specific enzyme.
17. 12. A method for producing a single-chain antibody or a labeled single-chain antibody, which comprises transcribing and translating the DNA according to the above 10 or 11 using a protein synthesis system.
18. The protein synthesis system is a wheat germ-derived cell-free protein translation system, and the concentration of the reducing agent in the translation reaction solution maintains the disulfide bond of the single-chain antibody to be produced and enables cell-free protein synthesis. The method for producing a single-chain antibody or a labeled single-chain antibody according to the above 16 or 17, wherein the concentration is the concentration.
19. The method for producing a single-chain antibody or a labeled single-chain antibody according to item 18, which is further carried out in the presence of an enzyme that catalyzes a disulfide bond exchange reaction.
20. A single-chain antibody or a labeled single-chain antibody having a Kd value equivalent to that of a natural type antibody produced by the method for producing a single-chain antibody or a labeled single-chain antibody according to the above item 19 using a wheat germ cell-free protein translation system. Chain antibody.
21. A solid-phased single-chain antibody characterized by contacting the antibody according to any one of the following with a substrate partitioned into a plurality of regions having a substance that specifically binds to a labeling substance of the antibody on the surface Production method.
1) A labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are crosslinked via a linker, and carrying a labeling substance on the linker portion.
2) In a labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker and carrying a labeling substance on the linker part, the heavy chain and the light chain of the antibody are variable regions. A labeled single-chain antibody characterized in that
3) A labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker and carrying a labeling substance on the linker part, wherein the labeling substance is present in the presence of a specific enzyme. A labeled single-chain antibody, which is a substance capable of binding to the polypeptide of the linker moiety of the antibody.
4) In a labeled single-chain antibody having a structure in which a heavy chain and a light chain, which are variable regions of an antibody, are crosslinked via a linker, and the linker moiety carries a labeling substance, the labeling substance is A labeled single-chain antibody, which is a substance capable of binding to a polypeptide of a linker portion of an antibody in the presence of an enzyme.
5) In a labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are crosslinked via a linker, and the linker moiety carries a labeling substance, the labeling substance is one of the linker parts of the antibody. A labeled single-chain antibody characterized by being incorporated as a part.
6) A labeled single-chain antibody having a structure in which a heavy chain and a light chain, which are variable regions of an antibody, are cross-linked via a linker, and a labeling substance is carried on the linker portion, wherein the labeling substance is an antibody A labeled single chain antibody characterized by being incorporated as part of a linker moiety.
7) The heavy chain and the light chain of the antibody have a structure in which they are crosslinked via a linker, and the linker part carries a labeling substance capable of binding to the polypeptide of the linker part of the antibody in the presence of a specific enzyme. A labeled single-chain antibody, wherein the labeling substance is biotin and the enzyme is biotin ligase.
8) Labeling having a structure in which the heavy and light chains that are the variable regions of the antibody are cross-linked via a linker and capable of binding to the polypeptide of the linker part of the antibody in the presence of a specific enzyme. A labeled single-chain antibody carrying a substance, wherein the labeled substance is biotin and the enzyme is biotin ligase.
22. 21. The method for producing a solid-phased single chain antibody as set forth in the preceding paragraph 21, which comprises immobilizing two or more different types of solid-phased single chain antibodies on a substrate divided into a plurality of regions. Method for producing single chain antibody.
23. 23. The production method according to item 21 or 22, wherein the labeling substance is biotin, and the substance that specifically binds to the labeling substance is streptavidin.
24. A solid-phase immobilized single-chain antibody prepared by the production method according to any of 21 to 23 above.
25. A method for analyzing an antigen-antibody reaction, which comprises contacting a test substance with the solid-phased single-chain antibody according to item 24 and analyzing the binding property with the solid-phased single-chain antibody.
26. A method for analyzing an antigen-antibody reaction, which comprises the following steps.
(1) a step of preparing a labeled single-chain antibody under conditions in which the disulfide bond of the single-chain antibody is retained, including the following elements (1) or (2):
(1) A DNA encoding a linker containing a nucleotide sequence capable of binding a labeling substance in the presence of a specific enzyme after translation, which is a DNA encoding a heavy chain and a light chain of an antibody capable of binding to a specific antigen. A step of producing a labeled single-chain antibody by transcribing the DNA ligated via the transcription-translation using a wheat cell-free protein synthesis system in the presence of a specific enzyme,
(2) A DNA containing a heavy chain and a light chain, which are variable regions of an antibody capable of binding to a specific antigen, has a linker containing a base sequence capable of binding a labeling substance in the presence of a specific enzyme after translation. A step of producing a labeled single-chain antibody by transcribing and translating the DNA linked via the encoding DNA using a wheat cell-free protein synthesis system in the presence of a specific enzyme,
(2) Prepare a substance (adapter substance) that specifically binds to the labeled substance of the labeled single-chain antibody when the labeled substance of the labeled single-chain antibody is a solid-phase substance, including the following elements Process,
(1) Immobilizing a substance (adapter substance) that specifically binds to the labeling substance of the labeled single-chain antibody on a substrate divided into a plurality of regions,
(2) A step of removing a substance (adapter substance) that specifically binds to the labeling substance of the labeled single-chain antibody that is not fixed to the substrate of (1) above,
(3) A step of appropriately removing non-specific adsorption on the substrate before and after the step (1) or (2) above,
(3) a step of preparing a solid-phased labeled single-chain antibody in the case where the labeled substance of the labeled single-chain antibody is a solid-phased substance, including the following elements:
(1) A substance that specifically binds the labeled substance of the labeled single-chain antibody prepared in (1) or (2) above to the labeled substance of the labeled single-chain antibody of (2) (adapter substance) ) A step of adding and contacting a necessary amount with a substrate divided into a plurality of areas having a surface,
(2) A step of removing the labeled single-chain antibody that is not fixed to the substance (adapter substance) that specifically binds to the labeled single-chain antibody on the substrate of (1) above,
(3) A step of removing non-specific adsorption on the substrate as appropriate, following the step of (2) in the first half,
(4) a step of preparing a labeled single-chain antibody in the case where the labeled substance is a signal substance, including the following elements:
(1) A step of appropriately removing non-specific adsorption on the substrate divided into a plurality of regions,
(2) A step of adding a necessary amount of the labeled substance of the labeled single-chain antibody prepared in (1) or (2) above to a substrate,
(5) A step of adding a required amount of a test substance to each substrate described in (3) or (4) above, and analyzing the binding property between the labeled single-chain antibody and the test substance,
(6) A step of qualitatively or quantitatively determining the interaction between the labeled single chain antibody and the test substance based on the binding result of (5).
27. A reagent kit for measuring an antigen-antibody reaction, which comprises a reagent used in the analysis method according to the above 25 or 26.
Is provided.

FIG. 1 is a diagram showing the structure of a translation template of the single chain antibody of the present invention.
FIG. 2 is an electrophoretic photograph showing the degree of biotin binding to a single-chain antibody by biotin ligase.
FIG. 3 is a diagram showing the degree of specific binding of the labeled single-chain antibody of the present invention to an antigen.
FIG. 4 is a diagram showing an association/dissociation curve between the labeled single-chain antibody of the present invention and an antigen.
FIG. 5 is a diagram showing the degree of binding between a single-chain antibody in which biotin is bound to a portion other than the linker portion and an antigen.
FIG. 6 is a diagram showing the binding degree of a single-chain antibody having a polyhistidine peptide in the linker portion to a nickel column.

(1) Single-chain antibody and labeled single-chain antibody
The single chain antibody used in the present invention is such that the heavy chain and the light chain of the antibody are bound via a linker and the antibody has an activity of binding to an antigen having a specific binding affinity. It may be any. Preferably, the antibody whose heavy chain is located at the N-terminus of the single-chain antibody molecule is used. The antibody is preferably a monoclonal antibody having an activity of recognizing and binding a specific antigen. Further, the heavy chain and light chain of an antibody do not need to include the entire length thereof, but may be any site sufficient for recognizing an antigen and having a specific affinity binding. Specifically, the variable region is preferably used.
The linker is not particularly limited as long as it has a length sufficient to crosslink the heavy chain and the light chain of the antibody through the linker and has a structure for having a labeling substance. Generally, a polypeptide consisting of 10 to 30 amino acids is preferably used. The specific structure can be appropriately selected according to the labeling substance described below.
As the labeling substance, those used for the purpose of labeling the single chain antibody of the present invention (hereinafter, this may be referred to as “signal substance”) and the purpose of immobilizing the single chain antibody of the present invention What is used (hereinafter, this may be referred to as “solid phase substance”) is preferable. Specifically, as the signal substance, a fluorescent dye capable of binding to an amino acid, such as a fluorescein series, a rhodamine series, an eosin series, an NBD series, or a photosensitizer, such as methylene blue or rose bengal, or a nucleus is used. Examples thereof include substances that give a specific signal in a magnetic resonance spectrum (NMR), such as amino acids containing a fluorine atom or a phosphorus atom. Further, the solid phase substance may be any substance as long as it is a substance that binds to a specific substance bound on the solid phase surface (hereinafter, this may be referred to as “adapter substance”). Examples of the combination of the immobilized substance and the adapter substance include biotin-binding proteins such as biotin/avidin and streptavidin, maltose/maltose binding proteins, guanine nucleotides/G proteins, polyhistidine peptides/metal ions such as nickel or cobalt, Glutathione-S-transferase/glutathione, DNA binding protein/DNA, antibody/antigen molecule (epitope), calmodulin/calmodulin binding peptide, ATP binding protein/ATP, or various receptor proteins such as estradiol receptor protein/estradiol/ligands thereof Etc. Any of these may be a solid phase substance or an adapter substance. Among these, it is preferable to use biotin as the solid phase substance and streptavidin as the adapter substance, or polyhistidine peptide as the solid phase substance and nickel as the adapter substance.
As the labeling substance, a substance capable of binding to the polypeptide of the linker portion of the antibody in the presence of a specific enzyme in the method of binding to the linker portion can also be used. Examples of such a substance include biotin. When biotin is used as the labeling substance, biotin ligase may be mentioned as the specific enzyme, and the linker may be one having an amino acid sequence recognized by biotin ligase.
Further, the labeling substance may be a substance incorporated as a part of the linker portion of the antibody, and a specific example thereof is a polyhistidine peptide. In this case, a linker containing a polyhistidine peptide is used.
The binding or incorporation of the labeling substance into the linker portion can be carried out by a method known per se depending on the properties of the signal substance or the solid-phased substance and the adapter substance used.
(2) Method for producing single-chain antibody and labeled single-chain antibody
The single chain antibody and the labeled single chain antibody of the present invention are produced, for example, by the following method. First, (i) a monoclonal antibody that recognizes a target protein or a part thereof as an antigen is produced, and (ii) a DNA encoding the monoclonal antibody is obtained. Furthermore, the sequences encoding the heavy chain and the light chain are specified, and the sequences encoding the linker are sandwiched between and linked (hereinafter, this DNA fragment may be referred to as “single chain antibody unit”). (Iii) The protein encoded by the produced single-chain antibody unit is synthesized by an appropriate method so that its structure is correctly retained. When the labeling substance is bound to the linker portion during or after the synthesis, this is bound. These detailed methods will be described below.
(I) Production of monoclonal antibody
The antigen of the single-chain antibody of the present invention is not particularly limited, and may be any one as long as it has immunogenicity. Specifically, for example, Salmonella sugar chains and the like can be mentioned. As a method for preparing a monoclonal antibody that specifically recognizes these antigens, a commonly used known method can be used, and a polypeptide used as an antigen also has a high antigenicity according to the known method, and an epitope (antigen determination). A suitable sequence can be selected and used as a group. As a method for selecting an epitope, commercially available software such as Epitope Adviser (manufactured by Fujitsu Kyushu System Engineering Co.) can be used.
As the polypeptide used as the above-mentioned antigen, it is preferable to use a synthetic peptide synthesized according to a known method. The polypeptide serving as an antigen may be prepared in a suitable solution or the like according to a known method to immunize a mammal such as rabbit or mouse, but the antigen may be used for stable immunization or to increase the antibody titer. It is preferable to use the peptide as a conjugate with an appropriate carrier protein or to immunize by adding an adjuvant or the like.
The route of administration of the antigen for immunization is not particularly limited, and any route such as subcutaneous, intraperitoneal, intravenous, or intramuscular may be used. Specifically, for example, a method of inoculating BALB/c mice with the antigen polypeptide several times every several days to several weeks is used. Further, the intake amount of the antigen is preferably 0.3 to 0.5 mg/once when the antigen is a polypeptide, but is appropriately adjusted depending on the type of polypeptide and the animal species to be immunized.
After immunization, appropriately test blood is collected to confirm an increase in antibody titer by a method such as a solid phase enzyme immunoassay (hereinafter, sometimes referred to as “ELISA method”) or Western blotting, and the antibody is sufficiently Blood is drawn from animals of increased value. By subjecting this to an appropriate treatment used for antibody preparation, a polyclonal antibody can be obtained. Specifically, there may be mentioned, for example, a method of obtaining a purified antibody obtained by purifying an antibody component from serum according to a known method. Alternatively, a monoclonal antibody can be produced by using a hybridoma obtained by fusing spleen cells of the animal and myeloma cells according to a known method (Milstein, et al., Nature, 256, 495 (1975)). .. The monoclonal antibody can be obtained, for example, by the following method.
First, antibody-producing cells are obtained from an animal whose antibody titer has been increased by immunization with the above-mentioned antigen. Antibody-producing cells are plasma cells and their precursor cells, lymphocytes, which may be obtained from any individual, but are preferably obtained from the spleen, lymph nodes, peripheral blood or the like. The myeloma to be fused with these cells is generally a cell line obtained from a mouse, for example, P3X63-Ag8.653 (ATCC:CRL) which is an 8-azaguanine resistant mouse (BALB/c-derived etc.) myeloma cell line. -1580), P3-NS1/1Ag4.1 (RIKEN Cell Bank: RCB0095) and the like are preferably used. For cell fusion, antibody-producing cells and myeloma cells are mixed at an appropriate ratio, and 50% polyethylene is added to an appropriate cell fusion medium such as RPMI1640, Iscove's modified Dulbecco's medium (IMDM), or Dulbecco's modified Eagle's medium (DMEM). It can be performed by using a solution in which glycol (PEG) is dissolved. It can also be performed by an electrofusion method (U. Zimmermann. et al., Naturewissenschaften, 68, 577 (1981)).
The hybridoma was prepared by using 5% CO in a normal medium (HAT medium) containing an appropriate amount of hypoxanthine/aminopterin/thymidine (HAT) solution by utilizing the fact that the myeloma cell line used was an 8-azaguanine resistant strain. _Two It can be selected by culturing at 37°C for an appropriate time. This selection method can be appropriately selected and used depending on the myeloma cell line to be used. By analyzing the antibody titer of the antibody produced by the selected hybridoma by the method described above, the hybridoma producing the antibody with a high antibody titer is separated by the limiting dilution method or the like, and the separated fused cells are cultured in an appropriate medium to obtain A monoclonal antibody can be obtained by purifying the obtained culture supernatant by an appropriate method such as ammonium sulfate fractionation or affinity chromatography. A commercially available monoclonal antibody purification kit can also be used for purification. In addition, ascites containing a large amount of the monoclonal antibody of the present invention can be obtained by growing the antibody-producing hybridoma obtained above in the abdominal cavity of an animal of the same strain as the immunized animal or a nude mouse.
(Ii) Acquisition of DNA encoding heavy chain and light chain of monoclonal antibody and preparation of single chain antibody unit
As a method for obtaining the DNA encoding the heavy chain (H chain) and the light chain (L chain) of the monoclonal antibody obtained in (i) above, specifically, an immunoglobulin obtained from a hybridoma producing the monoclonal antibody A part of the H chain and the L chain, preferably a part of the amino acid sequence of the amino acid sequence possessed by the variable region (V region), and a method of cloning a gene encoding the same based on the amino acid sequence, Can be mentioned. Here, the variable regions of the H chain and L chain of the monoclonal antibody are preferably composed of a framework region (FR) and a hypervariable region (CDR).
The DNA encoding the variable regions of the H and L chains thus obtained is, for example, as a single-chain antibody that recognizes the O-antigen of Salmonella, see Andand, N. et al. N. , Et al. J. Biol. Chem. , 266, 21874-21879 (1991).
A DNA encoding the linker is sandwiched between the DNAs encoding the variable regions of the H and L chains thus obtained, and both DNA fragments are ligated by an appropriate method to prepare a single chain antibody unit. Here, the single-chain antibody unit does not have to be obtained as a DNA fragment alone, and may be constructed at the same time as insertion into an expression vector or the like described later. The DNA encoding the linker may be any DNA as long as it encodes the linker described in (1). Specifically, for example, a DNA encoding a linker containing an amino acid sequence recognized by biotin ligase (Peter J. Schatz (1993) Biotechnology, 11 (1138-1143) is preferable, and examples thereof include those shown in SEQ ID NO: 1. Examples of the labeling substance incorporated as a part of the linker portion include those containing a base sequence encoding a polyhistidine peptide.
The DNA encoding the linker can be prepared by a method generally used, but it is preferably prepared by chemical synthesis.
(Iii) Production of single chain antibody
The single-chain antibody unit thus obtained is ligated under the control of an appropriate promoter and introduced into a host, or transcribed by an appropriate method and then produced using a cell-free protein translation system. A single-chain antibody can be produced by expressing the chain antibody under the condition that the disulfide bond of the chain antibody is retained. Here, an antibody having a low antigen-binding property can also be obtained as an antibody having a higher binding property by using an evolutionary engineering method known per se.
The appropriate promoter can be appropriately selected depending on the host used or the RNA synthase used for transcription. Specifically, when SP6 RNA synthase is used for transcription, it is preferable to use SP6 promoter. In addition, in the cell-free protein translation system, it is preferable to insert a nucleotide sequence that enhances translation activity between the promoter and the single-chain antibody unit. Specifically, in eukaryotes, 5′ cap structure (Shatkin, Cell, 9, 645-(1976)), Kozak sequence (Kizak, Nucleic Acid. Res., 12, 857- (1984)) and Shine-Dalgarno sequences are known in prokaryotes. Furthermore, it has been found that the 5'-untranslated leader sequence of RNA virus also has a translation promoting activity (Japanese Patent No. 2814433), and a method for efficiently performing protein synthesis using these sequences has been developed. (Japanese Patent Laid-Open No. 10-146197). In addition, a sequence obtained by a method of selecting a translation enhance sequence using an effect of the random sequence on polysome formation as an index can also be mentioned (Japanese Patent Application No. 2001-396941). The DNA thus produced may be hereinafter referred to as a translation template.
Specific examples of the translation template include those that recognize the O-antigen of Salmonella and have, for example, the structure shown in FIG.
As a host into which the translation template is introduced, a wheat germ-derived cell-free protein synthesis system, which is usually used for protein synthesis and is capable of retaining the disulfide bond of a single-chain antibody, is used. This is because the antibodies produced by other cell-free protein synthesis systems cannot sufficiently retain the three-dimensional structure for recognizing the antigen, and thus show only a low Kd value (ALEXANDER ZDANOV., et al., Proc. Natl. Acad. Sci. USA., Vol 91, pp. 6423-6427 (1994), C. Roger Mackenzie., et al., THE JOURNAL OF BIOLOGICAL CHEMISTRY., Vol 271, pp. 1527-1533. . Specifically, the wheat germ-derived cell extract used in the present invention is commercially available PROTEIOS. ^TM (Manufactured by TOYOBO) and the like.
Further, the reaction solution in which the intramolecular disulfide bond is retained and the protein is synthesized is a reaction solution for performing the above-mentioned wheat embryo-derived cell-free translation system (hereinafter, referred to as "weakly reduced translation reaction solution"). It can be prepared by adjusting the concentration of the reducing agent among the components necessary for protein synthesis (1). As a specific reducing agent and its concentration, dithiothreitol (hereinafter sometimes referred to as “DTT”) is used at a final concentration of 20 to 70 μM, preferably 30 to 50 μM, and 2-mercaptoethanol is added at a final concentration of 0.1. .About.0.2 mM, and the concentration of glutathione/oxidized glutathione is in the range of 30 to 50 .mu.M/1 to 5 .mu.M.
The concentration of the reducing agent in such a translation reaction solution is not limited to the above-mentioned one, and can be appropriately changed depending on the protein to be synthesized. The method of selecting the optimum concentration range of the reducing agent is not particularly limited, and examples thereof include a method of judging by the effect of an enzyme that catalyzes a disulfide bond exchange reaction. Specifically, a translation reaction solution having various reducing agent concentrations is prepared, and an enzyme that catalyzes a disulfide bond exchange reaction is added to these to synthesize a protein having a disulfide bond in the molecule. As a control experiment, similar protein synthesis is performed without adding an enzyme that catalyzes a disulfide bond exchange reaction to a similar translation reaction solution. The solubilized components of the protein synthesized here are separated by a method such as centrifugation. The solubilized component accounts for 50% or more of the total (solubilized rate 50%), and the reaction liquid in which the solubilized component is increased by the addition of the enzyme that catalyzes the disulfide bond exchange reaction is the intramolecular disulfide bond of the protein. It can be judged to be suitable as a reaction solution for synthesizing while retaining. Further, among the concentration ranges of the reducing agent selected by the effect of the enzyme that catalyzes the disulfide bond exchange reaction, the concentration of the reducing agent having the largest amount of synthesized protein can be selected as a more preferable concentration range. ..
As a method for adjusting a reaction solution having such a reducing agent concentration, a cell extract for wheat germ-derived cell-free protein synthesis containing no reducing agent is prepared, and the components necessary for the wheat germ-derived cell-free protein translation system are prepared using this. At the same time, a method of adding a reducing agent so that the concentration is within the above range, a method of removing the reducing agent from a cell extract for wheat germ-derived cell-free protein synthesis within the above concentration range, and the like are used. Since the cell extract for wheat germ-derived cell-free protein synthesis requires a high degree of reducing conditions when extracting it, the method of removing the reducing agent from this solution after extraction is simpler. Examples of the method for removing the reducing agent from the cell extract include a method using a carrier for gel filtration. Specifically, for example, a method in which a Sephadex G-25 column is preliminarily equilibrated with an appropriate buffer containing no reducing agent, and then the cell extract is passed through this, and the like can be mentioned.
Furthermore, after freeze-drying this cell extract to give a freeze-dried preparation, it is also possible to add an appropriate buffer to this and use it. When freeze-drying, the total concentration of the deliquescent substance is preferably 60 mM or less. It is also possible to add the above translation template to the cell extract and freeze-dry.
In the freeze-dried preparation, the content of the deliquescent substance (deliquescent material) that does not deteriorate the storage stability in the freeze-dried preparation is based on 1 part by weight of the protein contained in the freeze-dried preparation. 0.01 part by weight or less is preferable, and 0.005 part by weight or less is particularly preferable. The protein weight here is calculated by measuring the absorbance (260, 280, 320 nm).
The cell extract with the reducing agent concentration adjusted as described above may be hereinafter referred to as a weakly reduced translation reaction solution.
In addition, by adding an enzyme that catalyzes a disulfide bond exchange reaction to the weakly reduced translation reaction solution to carry out the translation reaction, a protein having an intramolecular disulfide bond can be synthesized with high efficiency. Examples of the enzyme that catalyzes the disulfide bond exchange reaction include protein disulfide isomerase. The amount of these enzymes added to the wheat germ-derived cell-free translation system can be appropriately selected depending on the type of enzyme. Specifically, when a protein disulfide isomerase is added to a translation reaction solution containing 20 to 70, preferably 30 to 50 μM of DTT as a reducing agent, which is a cell extract for cell-free protein synthesis extracted from wheat germ, The final concentration is in the range of 0.01 to 10 μM, preferably 0.5 μM. In addition, it is preferable to add it before the start of the cell-free translation reaction because of the efficiency with which disulfide bonds are formed.
In addition, examples of cell-free protein translation systems derived from plant seeds other than wheat include grasses such as barley, rice, and corn. However, among such cell-free protein translation systems, it is particularly preferable to use wheat germ extract, and the case of using this cell extract will be described as an example in detail.
As a method for selecting wheat germ, for example, Johnston, F. et al. B. et al. , Nature, 179, 160-161 (1957) can be used, and as a method for producing a cell extract from an embryo, Erickson, A. et al. H. et al. , Meth. In Enzymol. , 96, 38-50 (1996) and the like.
The wheat germ extract can be obtained by collecting the wheat germ extract and purifying it by gel filtration or the like according to the preparation method suitably used in the present invention. The gel filtration can be performed using a gel filtration device such as a Sephadex G-25 column. The composition and concentration of each component in the gel filtration solution are known per se, and those used in the method for producing a wheat germ extract for cell-free protein synthesis may be adopted. Here, as a solution for equilibrating a Sephadex G-25 column, a solution containing no reducing agent, specifically, for example, a solution containing HEPES-KOH, potassium acetate, magnesium acetate, or an L-type amino acid is used. By using it, about 97% of the reducing agent contained in the extract is absorbed. Specifically, when extraction is performed from wheat germ using an extract containing 1 mM DTT as a reducing agent, a wheat germ extract containing about 30 μM DTT can be finally obtained. However, since the activity of the wheat germ extract having a reduced concentration of the reducing agent is significantly reduced by cryopreservation, it is preferable to perform the step of removing the reducing agent immediately before using it in the translation reaction.
The germ extract after gel filtration may be contaminated with microorganisms, particularly spores of filamentous fungi (molds), and it is preferable to exclude these microorganisms. It is important to prevent the proliferation of microorganisms, especially during long-term (one day or more) cell-free protein synthesis reaction. The means for eliminating microorganisms is not particularly limited, but it is preferable to use a sterilizing filter for filtration. The pore size of the filter is not particularly limited as long as it is capable of removing microorganisms that may possibly be mixed, but usually 0.1 to 1 micrometer, preferably 0.2 to 0.5 micrometer is suitable. is there. By the way, since the size of the spores of Bacillus subtilis of a small class is 0.5 μm×1 μm, it is effective to use a 0.20 micrometer filter (for example, Minisart™ manufactured by Sartorius) to remove spores. .. In the filtration, it is preferable to first filter with a filter having a larger pore size, and then filter with a pore size capable of removing microorganisms that may possibly be mixed.
The cell extract thus obtained acts on substances (tritin, thionine, ribonuclease, etc., such as mRNA, tRNA, translated protein factor and ribosome) that suppress the protein synthesis function contained or retained by the raw material cells themselves. The endosperm containing the substance that suppresses its function) has been almost completely removed and purified. Here, the term "almost completely removed and purified of endosperm" means a wheat germ extract from which the endosperm portion has been removed to such an extent that the ribosomes are not substantially deadenated, and the ribosomes are substantially deadenized. The degree of not being adeninated means that the rate of denaturation of ribosomes is less than 7%, preferably 1% or less.
In addition, since the cell extract without such endosperm components contains a low-molecular weight protein synthesis inhibitor (hereinafter, this may be referred to as “low-molecular-weight synthesis inhibitor”), the cell extract It is preferable that these low molecular weight synthesis inhibitors are fractionated and eliminated from the constituent components according to the difference in molecular weight. The molecular weight of the substance to be excluded (low-molecular-weight inhibitory substance) may be smaller than the factor required for protein synthesis contained in the cell extract. Specific examples include those having a molecular weight of 50,000 to 14,000 or less, preferably 14,000 or less.
As a method for removing the low-molecular-weight synthesis inhibitor from the cell extract, a commonly used method known per se is used, and specifically, a method by dialysis through a dialysis membrane, a gel filtration method, or an ultrafiltration method. Law etc. are mentioned. Among these, the method by dialysis (dialysis method) is preferable in terms of easiness of supply of the substance to the dialysis solution. Hereinafter, the case of using the dialysis method will be described in detail as an example.
Examples of the dialysis membrane used for dialysis include those having an exclusion molecular weight of 50,000 to 12,000, specifically, a regenerated cellulose membrane having an exclusion molecular weight of 12,000 to 14,000 (Viskase Sales, manufactured by Chicago). Alternatively, Spectra/Pore 6 (manufactured by SPECTRUM LABORATORIES INC., CA, USA) having an exclusion molecular weight of 50,000 is preferably used. An appropriate amount of the above cell extract is placed in such a dialysis membrane and dialyzed by a conventional method. The time for dialysis is preferably about 30 minutes to 24 hours.
When insoluble components are produced in the cell extract when eliminating low-molecular-weight synthesis inhibitors, by inhibiting this (hereinafter, this may be referred to as "stabilization of the cell extract") The protein synthesis activity of the finally obtained cell extract (hereinafter this may be referred to as “post-treatment cell extract”) is increased. As a specific method of stabilizing the cell extract, a method of removing the low-molecular-weight inhibitor described above in a solution containing at least a high-energy phosphate compound such as ATP or GTP can be mentioned. ATP is preferably used as the high-energy phosphoric acid compound. Further, it is preferably carried out in a solution containing ATP and GTP, more preferably ATP, GTP, and 20 kinds of amino acids.
When eliminating low molecular weight inhibitors in a solution containing these components (hereinafter, this may be referred to as "stabilizing component"), after adding the stabilizing component to the cell extract and incubating, Alternatively, this may be subjected to a step of eliminating a low-molecular-weight inhibitor. When the dialysis method is used to eliminate the low-molecular weight synthesis inhibitor, the low-molecular weight inhibitor can be eliminated by adding a stabilizing component not only to the cell extract but also to the external solution of dialysis for dialysis. It is more preferable to add the stabilizing component to the external dialysis solution because a new stabilizing component is always supplied even if the stabilizing component is decomposed during dialysis. This can also be applied when using a gel filtration method or an ultrafiltration method, after equilibrating each carrier with a filtering buffer containing a stabilizing component, and then providing a cell extract containing the stabilizing component, Further, the same effect can be obtained by performing filtration while adding the above buffer solution.
The addition amount of the stabilizing component and the stabilizing treatment time can be appropriately selected depending on the type of cell extract and the preparation method. As a method for selecting these, a stabilizing component having a varied amount and type is experimentally added to the cell extract, and after a suitable time, a low molecular weight inhibitor elimination step is performed, and the obtained treated cell extract is extracted. Examples include a method in which a liquid is separated into a solubilized component and an insolubilized component by a method such as centrifugation, and the one having less insoluble component is selected. Furthermore, a method is also preferred in which cell-free protein synthesis is performed using the obtained treated cell extract and a protein with high protein synthesis activity is selected. Further, in the above selection method, when a cell extract and a dialysis method are used, an appropriate stabilizing component is also added to the external dialysate and dialyzed with these for an appropriate time, and then the obtained cell extract is obtained. There is also a method of selecting based on the amount of insoluble components therein, the protein synthesis activity of the obtained cell extract, and the like.
As an example of the stabilizing conditions of the cell extract thus selected, specifically, in the case of performing the step of eliminating the low-molecular-weight inhibitor by the dialysis method in the wheat germ extract prepared above, the wheat extract A method in which 100 μM to 0.5 mM of ATP, 25 μM to 1 mM of GTP, and 25 μM to 5 mM of 20 types of L-amino acids are added to the germ extract and the dialyzed external solution, and dialysis is performed for 30 minutes to 1 hour or more. Etc. The temperature for dialysis may be any temperature as long as protein synthesis activity is not lost and dialysis is possible. Specifically, the lowest temperature is a temperature at which the solution does not freeze, and is usually -10°C, preferably -5°C, and the highest temperature is 40°C, which is a temperature limit that does not adversely affect the solution used for dialysis. It is preferably 38°C.
The method of adding the stabilizing component to the cell extract is not particularly limited, and it is added before the step of eliminating the low-molecular-weight inhibitor and incubated for a suitable period of time to stabilize it. The exclusion step may be performed, or the removal step for the low-molecular-weight synthesis inhibitor may be performed using a cell extract containing a stabilizing component and/or a buffer solution used in the exclusion step containing a stabilizing component. May be.
The above-mentioned cell extract for cell-free protein synthesis is prepared in the concentration range of the reducing agent in the range described above, and the energy source, amino acid, translation template, tRNA, etc. necessary for cell-free protein synthesis, disulfide, etc. An enzyme that catalyzes a bond exchange reaction may be added as necessary and introduced into a selected system or apparatus known per se for protein synthesis. As a system or an apparatus for protein synthesis, a batch method (Pratt, JM et al., Transcription and Tranlation, Hames, 179-209, BD & Higgins, S. J., eds, IRL Press, Oxford (1984)), a continuous cell-free protein synthesis system (Spirin, AS et al., Science, 242, 1162-1164 (1988)) for continuously supplying an amino acid, an energy source and the like to a reaction system, The dialysis method (Kikawa et al., 21st Japan Society for Molecular Biology, WID6) or the multi-layer method (Sawasaki, T., et al., FEBS Let., 514, 102-105 (2002)) and the like can be mentioned.
Furthermore, a method of supplying a template RNA, an amino acid, an energy source, etc. to a synthesis reaction system when necessary, and discharging a synthesized product or a decomposed product when necessary (Japanese Patent Laid-Open No. 2000-333673: hereinafter referred to as "discontinuous gel"). Sometimes referred to as "filtration method") and the like can be used.
Of these, by using a continuous or discontinuous supply system of amino acids and energy sources, the reaction can be maintained for a long time and further efficiency can be achieved. When performing protein synthesis, it is preferable to use the batch method because the protein synthesis efficiency tends to be high. In addition, when the wheat germ extract is prepared by the method described above, it is not necessary to add it because it contains sufficient tRNA.
When protein synthesis is carried out by the batch method, it can be carried out, for example, by preincubating the synthesis reaction solution from which the translation template has been removed for an appropriate period of time, and then adding and incubating the translation template. As the synthesis reaction liquid, for example, 10 to 50 mM HEPES-KOH (pH 7.8), 55 to 120 mM potassium acetate, 1 to 5 mM magnesium acetate, 0.1 to 0.6 mM spermidine, and 0.025 each as a translation reaction liquid. ˜1 mM L-amino acid, 20 to 70 μM, preferably 30 to 50 μM DTT, 1 to 1.5 mM ATP, 0.2 to 0.5 mM GTP, 10 to 20 mM creatine phosphate, 0.5 to 1.0 U/μl Those containing RNase inhibitor, 0.01 to 10 μM protein disulfide isomerase, and 24-75% wheat germ extract are used.
When such a translation reaction solution is used, preincubation is performed at 10 to 40°C for 5 to 10 minutes, and incubation is also performed at 10 to 40°C, preferably 18 to 30°C, more preferably 20 to 26°C. The reaction time is the time until the reaction is stopped, but it is usually about 10 minutes to 7 hours in the batch method. (See Pratt, JM et al., Transcription and Translation, Hames, 179-209, BD & Higgins, SJ, eds, IRL Press, Oxford (1984)).
When protein synthesis is carried out by the dialysis method, protein synthesis is carried out using a device that is used as a dialysis inner solution for the synthesis reaction solution and is separated from the outer dialysis solution by a dialysis membrane capable of mass transfer (Kikawa et al. See Japan Society for Molecular Biology, WID6).
When the protein synthesis is carried out using the overlaying method, the synthesis reaction solution is placed in an appropriate container, and the dialyzed external solution described in the above dialysis method is overlaid on the solution so as not to disturb the interface. (Sawasaki, T., et al., FEBS Let., 514, 102-105 (2002), Patent Publication No. WO 02/24939 A1).
When protein synthesis is performed using the discontinuous gel filtration method, the synthesis reaction is performed using the synthesis reaction solution, and when the synthesis reaction is stopped, the template RNA, amino acid, energy source, etc. are supplied to synthesize or decompose the product. Protein synthesis is performed by discharging the substance. Specifically, for example, the above-mentioned synthesis reaction solution from which the translation template has been removed is pre-incubated for an appropriate period of time if necessary, then the translation template is added, and the mixture is placed in an appropriate container and reacted. Examples of the container include a microplate and the like. Under this reaction, for example, in the case of a reaction solution containing 48% by volume of wheat germ extract, the synthesis reaction is completely stopped within 1 hour of the reaction. This can be confirmed by measuring incorporation of amino acids into proteins and polyribosome analysis by sucrose density gradient centrifugation (Proc. Natl. Acad. Sci. USA., 97, 559-564 (2000)). The reaction solution in which the synthesis reaction has stopped is passed through a gel filtration column that has been equilibrated in advance with a feed solution having the same composition as the external dialysis solution described in the above dialysis method. By keeping the filtered solution at an appropriate reaction temperature again, the synthesis reaction is restarted and the protein synthesis proceeds for several hours. Hereinafter, this reaction and gel filtration operation are repeated. The reaction temperature and time are appropriately selected depending on the protein synthesis system to be used, but in the system using the wheat germ extract, it is preferable to repeat gel filtration at 26° C. about every hour.
In such cell-free protein translation, when a labeling substance is bound to the single-chain antibody of the present invention in the presence of a specific enzyme, the labeling substance and an enzyme capable of binding it to the polypeptide of the linker moiety The translation reaction described above is carried out in the presence of Specifically, when binding biotin as a labeling substance to a linker, biotin ligase (Avidity, LLC, etc.), which is an enzyme that recognizes an amino acid recognized by biotin ligase inserted into the linker in advance and binds biotin Translation reaction is performed in the presence of etc. The amount of biotin and biotin ligase added is preferably the amount described in the instructions attached to the commercial product (enzyme).
When the labeling substance is bound after the protein synthesis, it may be bound to the linker portion of the single-chain antibody in the translation reaction solution after the translation reaction by a method suitable for each labeling substance. Alternatively, the single chain antibody may be purified by the following method and then bound by a method suitable for each labeling substance.
The thus-obtained single-chain antibody or labeled single-chain antibody of the present invention can be confirmed by a method known per se. Specifically, for example, incorporation of amino acids into proteins, separation by SDS-polyacrylamide gel electrophoresis and staining with Coomassie Brilliant Blue (CBB), autoradiography method (Endo, Y. et al., J. Biotech. , 25, 221-230 (1992); Proc. Natl. Acad. Sci. USA., 97, 559-564 (2000)) and the like can be used.
Further, since the reaction solution thus obtained contains the target single-chain antibody or the labeled single-chain antibody in a high concentration, it is known per se such as dialysis, ion exchange chromatography, affinity chromatography, gel filtration and the like. The desired single-chain antibody or labeled single-chain antibody can be easily obtained from the reaction solution by a separation and purification method.
(3) Use of labeled single chain antibody
The labeled single-chain antibody of the present invention can be used in a method for analyzing an antigen-antibody reaction by analyzing the binding property with an antigen. The analysis method of the antigen-antibody reaction can be performed by including the following steps (I) to (VI).
(I) a step of preparing a labeled single-chain antibody under conditions in which the disulfide bond of the single-chain antibody is retained, including the following elements (1) or (2):
(1) A DNA encoding a linker containing a nucleotide sequence capable of binding a labeling substance in the presence of a specific enzyme after translation, which is a DNA encoding a heavy chain and a light chain of an antibody capable of binding to a specific antigen. A step of producing a labeled single-chain antibody by transcribing the DNA ligated via the transcription-translation using a wheat cell-free protein synthesis system in the presence of a specific enzyme,
(2) A DNA containing a heavy chain and a light chain, which are variable regions of an antibody capable of binding to a specific antigen, has a linker containing a base sequence capable of binding a labeling substance in the presence of a specific enzyme after translation. A step of producing a labeled single-chain antibody by transcribing and translating the DNA linked via the encoding DNA using a wheat cell-free protein synthesis system in the presence of a specific enzyme,
(II) Prepare a substance (adapter substance) that specifically binds to the labeled substance of the labeled single-chain antibody when the labeled substance of the labeled single-chain antibody is a solid-phase substance, including the following elements Process,
(1) Immobilizing a substance (adapter substance) that specifically binds to the labeling substance of the labeled single-chain antibody on a substrate divided into a plurality of regions,
(2) A step of removing a substance (adapter substance) that specifically binds to the labeling substance of the labeled single-chain antibody that is not fixed to the substrate of (1) above,
(3) A step of appropriately removing non-specific adsorption on the substrate before and after the step (1) or (2) above,
(III) a step of preparing a solid-phase immobilized single-chain antibody when the labeling substance is a solid-phase immobilized substance, including the following elements:
(1) A plurality of substances each having a substance (adapter substance) capable of specifically binding the labeled single chain antibody prepared in (1) or (2) above to the labeled single chain antibody according to (II) on the surface The step of adding and contacting the necessary amount to the base divided into the area
(2) A step of removing the labeled single-chain antibody that is not fixed to the substance (adapter substance) that specifically binds to the labeled single-chain antibody on the substrate of (1) above,
(3) Following the steps of (2) in the previous term, a step of removing non-specific adsorption on the substrate as appropriate,
(IV) a step of preparing a labeled single-chain antibody when the labeled substance is a signal substance, which comprises the following elements,
(1) A step of appropriately removing non-specific adsorption on the substrate divided into a plurality of regions,
(2) A step of adding a necessary amount of the labeled substance of the labeled single-chain antibody prepared in (1) or (2) above to a substrate,
(V) adding a required amount of the test substance to each substrate described in (III) or (IV), and analyzing the binding property between the labeled single chain antibody and the test substance,
(VI) A step of qualitatively or quantitatively determining the interaction between the labeled single-chain antibody and the test substance based on the binding result of (V).
In the above-mentioned antigen-antibody analysis method, the conditions under which the disulfide bond of the single-chain antibody is retained may be the conditions under which the disulfide bond of the labeled single-chain antibody produced can be retained in the step of producing the labeled single-chain antibody. There is no particular limitation. Specifically, it can be performed by adjusting the concentration of the reducing agent in the translation reaction solution, which is the method described in (iii) Production of single chain antibody. Further, the method of removing the adapter substance and the labeled single-chain antibody is to remove the substrate from the substrate by washing the substrate several times with a washing buffer usually used by those skilled in the art. In addition, the method of removing non-specific adsorption in the adapter substance fixed to the substrate is to fill the substrate with a booking solution or the like usually used by those skilled in the art. Then, it can be performed by washing with a buffer solution several times.
When the single chain antibody is immobilized, a method well known in the art can be used to reduce non-specific adsorption. Specifically, it includes a method of pre-coating an array solid support with bovine serum albumin (BSA), reduced low-fat milk, salmon sperm DNA, porcine heparin, etc. (Ausubel,. et al., Short Protocols in Molecular Biology). , 3rd. edition (1995)).
As a base used in the above-described antigen-antibody analysis method, a base suitable for an antigen-antibody reaction analysis method or apparatus can be used. Specifically, when an enzyme-linked immunosorbent assay (Enzyme Linked Immunosorbent Assay (ELISA): Crowthjer, JR, Methods in Molecular Biology, 42, (1995)) is used, it is usually used by the ELISA method. Plastic microtiter plates are preferred. When the surface plasmon resonance method (Cullen, D.C. et al., Biosciences, 3(4), 211-225 (1987-88)) is used, gold, silver, platinum, etc. are formed on a transparent substrate such as glass. The metal thin film is preferably formed. When an evanescent field molecular imaging method (Funatsu, T., et al., Nature, 374, 555-559 (1995)) is used, a transparent body such as glass is preferable, and a quartz glass is more preferable. Is used. When the fluorescence imaging analysis method is used, a nitrocellulose membrane or nylon membrane, which is usually used for immobilizing proteins and the like, or a plastic microtiter plate can also be used. Further, complex sugars (eg, agarose and sepharose), acrylic resins (eg, polyacrylamide and latex beads), magnetic beads, silicon wafers, etc. can also be used as a substrate.
For the attachment of the adapter substance to such a substrate, a commonly used method known per se can be used. Specifically, diazo method, peptide method (method using acid amide derivative, carboxychloride resin, maleic anhydride derivative, inocyanate derivative, cyanogen bromide activated polysaccharide, cellulose carbonate derivative, etc., alkyl method, crosslinking reagent Examples thereof include a method used, a method by Ugi reaction, etc. Further, when a substrate such as glass is used, a method of physically adsorbing is also used. Furthermore, a commercially available method such as streptavidin-magnet (manufactured by Promega) is used. It is also possible to use the one.
The labeled single-chain antibody thus obtained is brought into contact with a solution containing one or more known antigens and other test substances, and an antigen-antibody reaction is analyzed to obtain an antibody having binding specificity to the antigen. Can be identified. The antigen may be a protein, an organic compound, a carbohydrate, a nucleic acid or the like. These may be isolated or may be recombinant or naturally occurring. The amount of antigen used is preferably in the range of about 1-100 ng/μl. The time required for the antigen-antibody reaction is usually in the range of 5 minutes to 24 hours, and generally 0.5 to 2 hours is preferable.
After the antigen-antibody reaction, in the case of a solid-phase immobilized single-chain antibody, a step of washing the solid phase to which the antibody is bound with a biochemical buffer containing a surfactant and the like can be added. The composition of the buffer solution, the number of washings, etc. are appropriately selected depending on the strength of the antigen-antibody reaction and the like.
When the labeled substance is a solid phase substance, the binding method between the solid phase immobilized single chain antibody and the antigen is analyzed, and the labeled substance is a signal substance. Can analyze the antigen-antibody reaction by analyzing the binding property with the antigen in a solution.
The method for quantitatively or qualitatively determining the interaction between the labeled single-chain antibody and the test substance can be carried out by a commonly used method known per se. Specific examples include an ELISA method, a surface plasmon resonance method, an evanescent field molecular imaging method, a fluorescence imaging analysis method, and a method using a radioisotope label.
The test substance such as an antigen may be any substance that may contain an antigen. Specific examples include body fluids such as blood, cell wall extracts of bacteria, and protein mixtures.
According to the method for analyzing an antigen-antibody reaction using a labeled single-chain antibody of the present invention and the reagent kit for measuring an antigen-antibody reaction containing a reagent in the analysis method, for example, the presence or absence of human autoantibodies or cancer cells It can be a tool for analyzing and diagnosing specific antigens and the like.

比較例１ ビオチンの結合位置による固相化効率の検討
単鎖抗体への化学結合によるビオチンの付加
本方法は、続生化学実験講座、免疫生化学研究法（日本生化学会、東京化学同人（１９８６））の抗体標識法に記載の方法を用いた。
実施例１に記載の方法で、翻訳反応時にビオチンリガーゼ及びビオチンを添加しないで合成した反応液を、１５，０００ｒｐｍ、１０分間の遠心を行い、上清を取得した。上清の可溶画分２５μｌを同量の１Ｍ炭酸水素ナトリウム溶液で希釈した後、Ｇ−２５セファデックスカラムにより緩衝液を交換し、１μｌのＮＨＳ−ｂｉｏｔｉｎ（Ｎ−ｈｙｄｒｏｘｙｓｕｃｃｉｍｉｄｅ−ｂｉｏｔｉｎ、５０ｍｇ／ｍｌ ＤＭＳＯ）を添加した。これを４℃で一晩反応させた後、以下に示すように抗原との結合性を解析した。
抗原との結合活性の解析
上記（１）で調製した反応液３０μｌを１０ｍＭ ＰＢＳＴ（ｐＨ８．０）、０．６ｍＭ ＣａＣｌ_２で平衡化したＧ−２５ ｓｐｉｎ ｃｏｌｕｍｎにより過剰のビオチンをゲルろ過した。そのタンパク質溶液４０μｌを予め０．６ｍＭ ＣａＣｌ_２を含むコムギ胚芽抽出液２５μｌで洗浄しておいた１０μｌの上記実施例２（１）で作製した固相化サルモネラ糖鎖抗原（Ｓａｌ−Ｍａｇ）と共に９６ｗｅｌｌマイクロプレート上に添加した。１５分間穏やかに混合した後、４０μｌの０．１５Ｍ ＮａＣｌ／１０ｍＭ ＰＢＳＴ（ｐＨ８．０）で４回洗浄し、最後に同量の０．１Ｍ ｇｌｙｃｉｎｅ−ＨＣｌ（ｐＨ２．４）で４回溶出を行った。この結果を図５に示す。活性の保持した抗体であれば、後半の酸性緩衝液により溶出されるはずであるが、図から明らかなように、フラクション番号１０〜１３にはタンパク質の存在はみられず、１番目の素通り画分に大半が存在した。このことは、上記の化学的方法で製造したビオチン化単鎖抗体は抗原結合活性を失っていることを示している。
実施例３ ポリヒスチジンペプチドを挿入した単鎖抗体の製造および固相化
（１）ポリヒスチジンペプチドをリンカー部分に含む単鎖抗体の製造
実施例１（１）に記載のｓｃｆｖ−ｐＥＵを鋳型として、配列番号８および９に記載の塩基配列からなるプライマーによりＬＡ ｔａｑ（ＴＡＫＡＲＡ社製）キットを用いてＰＣＲを行った。ＰＣＲ反応液は、５μｌ １０×ＬＡ ｂｕｆｆｅｒ、５μｌ ２５ｍＭ塩化マグネシウム、８μｌ ２．５ｍＭ ｄＮＴＰ、各１μｌ ２０μＭプライマー、０．１ｎｇ鋳型プラスミド／５０μｌに調製し、９４℃１分×１サイクル、９４℃４５秒／５５℃１分／７２℃１分３０秒×３０サイクル、７２℃５分の反応を行った。増幅されたＤＮＡ断片は、常法に従い、ＫＯＤ Ｔ４ ｐｏｌｙｍｅｒａｓｅ（ＮＥＢ社製）により末端の平滑化を行ったのち、Ｐｏｌｙｎｕｃｌｅｏｔｉｄｅ ｋｉｎａｓｅ（ＮＥＢ社製）によるリン酸化後、Ｌｉｇａｔｉｏｎ ｈｉｇｈ（東洋紡社製）によりＳｅｌｆ Ｌｉｇａｔｉｏｎを行い、環状のプラスミド（図１：以下、これを「ｓｃＦＶ−ｐＨＩＳ−ｐＥＵ」と称することがある）を作製した。
このプラスミドを鋳型として、実施例１（３）に記載の方法により転写し、ｍＲＮＡを精製した後に、翻訳反応液中のＤＴＴを２００μＭのメルカプトエタノールに換えて翻訳反応を行った。翻訳反応３時間後の反応液を１５，０００ｒｐｍ、１０分間の遠心分離によって可溶化成分を分離し、過剰量のメルカプトエタノールを５０ｍＭリン酸緩衝液（ｐＨ７．０）、５００ｍＭ ＮａＣｌ、５％ Ｇｌｙｃｅｒｏｌ（Ｂｉｎｄｉｎｇ ｂｕｆｆｅｒ）で平衡化したＧ−２５スピンカラムにより除去した。
スピンカラムの溶出液、２０μｌを同量のＢｉｎｄｉｎｇ ｂｕｆｆｅｒで希釈後、予めｂｉｎｄｉｎｇ ｂｕｆｆｅ １５０μｌで６回洗浄したニッケルカラム（Ｍｅｔａｌ ａｆｆｉｎｉｔｙ ｒｅｓｉｎ：ＴＡＬＯＮ社製）２００μｌ（５０％ ｒｅｓｉｎ）に８０μｌ添加し、室温で１時間インキュベートした。このカラムを１５０μｌの５０ｍＭリン酸緩衝液（ｐＨ７．０）、５００ｍＭ ＮａＣｌ、５％ Ｇｌｙｃｅｒｏｌ、６ｍＭ Ｉｍｍｉｄａｚｏｌｅ（ｗａｓｈｉｎｇ ｂｕｆｆｅｒ）で４回洗浄し（図中ｗ１〜ｗ４）、さらに１５０μｌの５０ｍＭリン酸緩衝液（ｐＨ７．０）、５００ｍＭ ＮａＣｌ、１５０ｍＭ Ｉｍｍｉｄａｚｏｌｅ（ｅｌｕｔｉｏｎ ｂｕｆｆｅｒ）で５回溶出（図中ｅ１〜ｅ５）を行った。各フラクションに含まれる単鎖抗体の量は、１４Ｃ ｄｐｍ値により測定した。この結果を図６に示す。図中、Ｃは、カラムにかける前のタンパク質含有液の全量中の１４Ｃ ｄｐｍ値を示す。グラフの横軸は、フラクション番号を示し、ｗ１〜ｗ４は、ｗａｓｈｉｎｇ ｂｕｆｆｅｒにより溶出されたフラクション中の、またｅ１〜ｅ５はｅｌｕｔｉｏｎ ｂｕｆｆｅｒにより溶出されたフラクション中の１４Ｃ ｄｐｍ直を示す。ＥＴはｅ１〜ｅ５中の１４Ｃ ｄｐｍ値の合計を示す。
フラクション番号ｅ１〜ｅ５は、ニッケルと特異的に結合するポリヒスチジンペプチドを含む単鎖抗体の存在を示している。図から明らかなように、全合成量の約５０％近くの単鎖抗体は、ニッケルカラムにより精製できることがわかった。精製した単鎖抗体は、抗原結合活性を保持していることも実施例２に記載の方法により確認できた。この結果は、ポリヒスチジンペプチドをリンカー部分に組み込んだ単鎖抗体は、ニッケル固相上に活性を保持した状態で固定化しうることを示している。Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.
Example 1 Production of biotinylated anti-Salmonella single-chain antibody (1) Preparation of DNA encoding Salmonella single-chain antibody and linker Anti-Salmonella single-chain antibody was selected as the single-chain antibody of the present invention, and the following experiment was conducted. It was The X-ray three-dimensional structure of the antibody has already been analyzed, and molecular recognition of sugar chains has been investigated in detail (Cygler, M., et al., Science, 253, 442-445 (1991); Bundle, D.R. , Et al, Biochemistry, 33, 5172-5182 (1994)). Lipopolysaccharide is present on the cell surface of Salmonella bacteria, and anti-Salmonella antibody binds to the most extracellular O-antigen of this lipopolysaccharide (Andand, NN, et al., Protein Engin. , 3, 541-546 (1990)). There is a report that a single chain antibody in which a VL chain and a VH chain, which are antigen recognition sites that specifically bind to the O-antigen, are linked by a specific linker is expressed in Escherichia coli in large quantities (Anand, NN, et al., J. Biol. Chem., 266, 21874-21879 (1991)). In order to synthesize a single-chain antibody in an active state, formation of a disulfide bond existing in each of the VL chain and the VH chain is indispensable (Zdanov, ALY, et al., Proc. Natl. Acad.Sci.USA., 91, 6423-6427 (1994)), and thus the single-chain antibody was the subject of the method of the present invention.
The DNA encoding the anti-Salmonella single-chain antibody is a plasmid containing a DNA encoding the single-chain antibody against the wild-type Salmonella O-antigen (Anand, NN, et al., J. Biol. Chem., 266. 21874-21879 (1991)) as a template, and the polymerase chain reaction (PCR) was performed using the primers consisting of the nucleotide sequences of SEQ ID NOs: 2 and 3. The obtained DNA fragment was inserted into a pGEMT-easy vector (manufactured by Promega), and treated with BglII and NotI. The obtained DNA fragment was inserted into a pEU vector which had been previously treated with the same restriction enzymes. PCR was carried out using this plasmid as a template and primers having the nucleotide sequences of SEQ ID NOs: 4 and 5 to introduce a stop codon. The plasmid prepared here is called scfv-pEU.
Next, a DNA having the DNA sequence (SEQ ID NO: 1) encoding the biotin ligase recognition sequence inserted into the linker portion was prepared. First, PCR was carried out using the above-prepared plasmid scfv-pEU as a template and a primer composed of the nucleotide sequences of SEQ ID NOs: 6 and 7 using an LA Taq (manufactured by TAKARA) kit. The PCR reaction solution was prepared in 5 μl 10×LA buffer, 5 μl 25 mM magnesium chloride, 8 μl 2.5 mM dNTP, 1 μl each 20 μM primer, 0.1 ng template plasmid/50 μl, 94° C. 1 min×1 cycle, 94° C. 45 sec. /55° C. 1 minute/72° C. 1 minute 30 seconds×30 cycles, 72° C. 5 minutes. The amplified DNA fragment was blunt-ended with KOD T4 polymerase (manufactured by NEB) according to a conventional method, then phosphorylated with Polynucleotide kinase (manufactured by NEB), and then Self Ligation by Ligation High (manufactured by Toyobo). Then, a circular plasmid (FIG. 1: hereinafter, this may be referred to as “scFv-biotin-pEU”) was prepared.
(2) Preparation of a cell extract for weakly reducing type cell-free protein synthesis Hokkaido seed wheat seeds (undisinfected) were added to a mill (Frotsch: Rotor Speed mill pulverisette 14 type) at a rate of 100 g per minute, and the rotation speed was adjusted. The seeds were gently ground at 8,000 rpm. After collecting a fraction (mesh size 0.7 to 1.00 mm) containing germs having germination ability by sieving, a mixed solution of carbon tetrachloride and cyclohexane (volume ratio=carbon tetrachloride:cyclohexane=2.4:1). ) Was used to collect the floating fraction containing germs with germination ability, remove the organic solvent by drying at room temperature, and remove the impurities such as seed coat mixed by blowing air at room temperature to obtain the crude embryo fraction. Obtained.
Next, using a belt type color sorter BLM-300K (manufacturer: Anzai Manufacturing Co., Ltd., distributor: Anzai Sogyo Co., Ltd.), the embryos were selected from the crude embryo fraction by utilizing the difference in color as follows. .. This color sorter has a means for irradiating the crude germ fraction with light, a means for detecting reflected light and/or a transmitted light from the crude germ fraction, a means for comparing a detected value with a reference value, and a deviation from the reference value. It is a device having a means for selecting and excluding objects or those within a reference value.
The crude germ fraction was supplied onto a beige belt of a color sorter at a rate of 1000 to 5000 grains/cm ^2, and the crude germ fraction on the belt was irradiated with light by a fluorescent lamp to detect reflected light. .. The belt conveyance speed was 50 m/min. A monochrome CCD line sensor (2048 pixels) was used as a light receiving sensor.
First, a standard value was set between the brightness of the embryo and the brightness of the seed coat in order to eliminate components darker in color than the embryo (seed coat, etc.), and those deviating from the standard value were removed by suction. Next, in order to select the endosperm, a standard value was set between the brightness of the germ and the brightness of the endosperm, and those outside the standard value were removed by suction. Suction was performed using 30 suction nozzles (one suction nozzle per 1 cm length) arranged at a position approximately 1 cm above the conveyor belt.
By repeating this method, the germs were selected until the purity of the germs (the weight ratio of the germs contained in 1 g of an arbitrary sample) reached 98% or more.
The obtained wheat germ fraction was suspended in distilled water at 4° C. and washed with an ultrasonic washing machine until the washing liquid did not become cloudy. Then, the suspension was suspended in a 0.5% by volume solution of Nonidet (Nakarai Tectonics) P40, and washed with an ultrasonic washing machine until the washing solution did not become cloudy to obtain wheat germ. Performed at °C.
2 volumes of extraction solvent (80 mM HEPES-KOH (pH 7.8), 200 mM potassium acetate, 10 mM magnesium acetate, 8 mM dithiothreitol, (0.6 mM each of 20 types of L-type amino acids) with respect to the washed embryo wet weight. )) was added, and the embryos were subjected to limited crushing three times at 5,000 to 20,000 rpm for 30 seconds each three times using a Waring blender. From this homogenate, a centrifugation supernatant obtained by centrifugation at 30,000 xg for 30 minutes using a high speed centrifuge was centrifuged again under the same conditions to obtain a supernatant. This sample showed no decrease in activity after long-term storage at -80°C or lower. The obtained supernatant was passed through a filter (New Stella Disc 25: manufactured by Kurashiki Spinning Co., Ltd.) having a pore size of 0.2 μM to perform filter sterilization and removal of mixed fine dust.
Next, this filtrate was previously prepared as a solution (40 mM HEPES-KOH (pH 7.8), 100 mM potassium acetate, 5 mM magnesium acetate, 20 mM L-type amino acid mixture of 0.3 mM each (depending on the purpose of protein synthesis, The gel filtration was performed using a Sephadex G-25 column that had been equilibrated with (does not need to be added or may be a labeled amino acid)). The obtained filtrate was again centrifuged at 30,000×g for 30 minutes, and the concentration of the collected supernatant was adjusted to A260 nm of 90 to 150 (A260/A280=1.4 to 1.6). It was stored at -80°C or lower until it was used for the dialysis treatment or protein synthesis reaction described below.
(3) Protein synthesis using weakly reduced translation reaction solution (when biotin and biotin ligase were added during translation)
The translation template DNA obtained in (1) above was transcribed using SP6 RNA polymerase (manufactured by TOYOBO). As a reaction solution, 80 mM HEPES-KOH (pH 7.6), 16 mM magnesium acetate, 2 mM spermidine, 10 mM DTT, NTPs each 2.5 mM, 0.8 U/μl RNase inhibitor, 50 μg/ml plasmid, and 1.2 U/μl 400 μl of SP6 RNA polyMerase/ddw was used. After incubating at 37° C. for 2 hours, phenol/chloroform extraction and purification by NICK column (manufactured by Amersham Pharmacia) were performed. After ethanol precipitation, the precipitate was dissolved in 35 μl of purified water.
A translation reaction was performed using the obtained mRNA. The translation reaction solution was 1.2 mM ATP, 0.25 mM GTP, 15 mM creatine phosphate, 0.4 mM spermidine, 29 mM HEPES-KOH (pH 7.6), 95 mM potassium acetate, 2.7 mM magnesium acetate, 0.23 mM L type. Amino acid, 0.58 U/μl RNase inhibitor (manufactured by Promega), 4 nCi/μl 14C-Leu, 7.5 μg mRNA, 0.5 μM PDI, 19.5 μM biotin (Nacalai), 19.5 μg/μl biotin ligase (avidity) The reaction was performed by a batch method at 26° C. for 3 hours with a 12 μl wheat germ extract. As a control, a translation reaction without addition of biotin was also performed.
The reaction solution after 3 hours of the translation reaction was centrifuged at 15,000 rpm for 10 minutes to separate the solubilized components, and the remaining unreacted biotin was equilibrated with 50 mM Tris (pH 8.0) using a G-25 spin column. Removed. 20 μl of the eluate from the spin column was diluted with the same amount of 50 mM Tris (pH 8.0) buffer, 5 μl of streptavidin magnetic beads (manufactured by Promega) was added, and the mixture was gently mixed at room temperature. After collecting the magnetic beads with a magnetic field, a supernatant fraction was obtained, separated by SDS-PAGE, and the amount of anti-Salmonella single chain antibody was measured by autoradiography. This result is shown as co-transl. It is shown in biotinylation. As is clear from the figure, most of the translations performed in the presence of biotin and biotin ligase (in the figure: +biotin) were recovered from the magnetic beads by binding with streptavidin, and biotin was added in reverse. It was shown that there was almost no antibody bound to the magnetic beads in the case of not performing (-biotin). From this, it was revealed that biotin was bound to the anti-Salmonella single chain antibody by the method described above.
(4) Protein synthesis using weakly reduced translation reaction solution (when biotin and biotin ligase are added after the translation reaction)
Similar to the method for producing an anti-Salmonella single chain antibody described in (1) to (3) above, the results obtained by adding biotin and biotin ligase 3 hours after the initiation of the translation reaction were the results of post-transl. It is shown in biotinylation. As is clear from the figure, it was found that the amount of biotinylated antibody removed from the magnetic beads was almost absent in the one with biotin added (+) and the one without biotin (-). From this, it was found that the biotin ligase and biotin are preferably added during the translation reaction.
Example 2 Immobilization of biotinylated single-chain antibody and analysis of antigen-antibody reaction (1) Preparation of aldehyde-modified Salmonella O-antigen Lipopolysaccharide (manufactured by SIGMA) 20 mg (2.8 μmol) was added to 0.25 M sodium hydroxide. It was dissolved in 20 μl of an aqueous solution and stirred at 56° C. for 1 hour. After dialysis against distilled water, 200 mg (0.8 mmol) of sodium metaperfolate was added, and the mixture was stirred for 5 minutes in the dark. After further adding 1 ml of ethylene glycol and stirring for 1 hour, this was dialyzed against distilled water and freeze-dried to obtain an aldehyde-type Salmonella sugar chain powder. This was dissolved in 0.2 ml of 20 mM sodium borate buffer pH 9.0 (10 mg/ml). Aminated magnetic beads (NH2-Mag: manufactured by Polyscience) 0.1 ml were washed three times with 0.4 ml of the same buffer solution to equilibrate, and then added to the above aldehyde-type Salmonella sugar chain solution, and allowed to stand at room temperature for 6 hours. The reaction was carried out. The magnetic beads were washed 3 times with 0.4 ml of the same buffer. The immobilization rate of sugar chains on the magnetic beads was determined by quantifying sugar chains remaining in the supernatant by the phenol/sulfuric acid method. Here, the binding rate of the sugar chain to the magnetic beads was 40% (0.13 μmol Salmonella sugar chain/100 μl magnetic beads).
(2) Binding of Biotinylated Anti-Salmonella Single-Chain Antibody to Salmonella Sugar Chain After synthesizing the biotinylated single-chain antibody by the method described in Example 1 (total 38 μl), 10 mM PBST (pH 8.0), 0.6 mM Excess biotin was gel filtered through a G-25 spin column equilibrated with CaCl ₂ . 40 μl of the protein solution was washed with 25 μl of wheat germ extract containing 0.6 mM CaCl ₂ in advance and added to a 96-well microplate together with 10 μl of the immobilized Salmonella antigen (Sal-Mag) prepared in (1) above. did. After gently mixing for 15 minutes, the plate was washed 4 times with 40 μl of 0.15 M NaCl/10 mM PBST (pH 8.0), and finally eluted with the same amount of 0.1 M glycine-HCl (pH 2.4) 4 times. It was The first wash elutes the single chain antibody that did not bind to the antigen, and the subsequent elution elutes the single chain antibody that bound to the antigen. The amount of protein in each fraction (5 μl) was determined by 14C counting. The result is shown in FIG. Here, the binding result when the mutant G102D was similarly biotinylated for the purpose of confirming that the biotinylated single chain antibody retains the antigen specificity is also shown. As is clear from the figure, in the case of the wild type (Wild type), the active fraction (no. 6) eluted in the acidic pH solution is present in nearly 50% of the total amount of the antibody, whereas that of the mutant G102D. In this case, no active fraction was present and most of the no. It appears in the 1st pass fraction. This result shows that the biotinylated single-chain antibody retains the original antigen-binding activity, and it is supported that co-translational biotinylation proceeds without impairing the antigen-binding activity at all. It was
(3) Measurement of dissociation equilibrium constant using biomolecular interaction analyzer (Iasys) First, streptavidin (0.1 mg/ml: manufactured by Nakarai Co., Ltd.) was immobilized (immobilized) on a biotin cuvette (manufactured by Affinity Sensors). Amount: 674 arc sec., 27.2 ng, 0.97 pMol). Next, the biotinylated single chain antibody prepared in Example 1 was purified by using the immobilized Salmonella sugar chain antigen (Sal-Mag) by the method of (2) above. From the 14C dpm value, 8.4 pmol/50 μl of purified biotinylated single chain antibody was obtained. This 50 μl portion was added to the cuvette and immobilized on streptavidin (immobilization amount: 433.6 arc sec, 11.5 ng, 0.4 pmol). The association and dissociation curves were measured by adding various concentrations of Salmonella sugar chains (2.4, 4.8, 9.7, 12.9, 19.4 μM) thereto. The results are shown in FIG. 4, and the dissociation equilibrium constant obtained from the curve is shown in Table 1. In addition, in Table 1, the values obtained by a single-chain antibody synthesized using live E. coli cells by the same method (MacKenzie, CR et al., J. Biol. Chem., 271, 1527-1533 (1996). )) is also shown for comparison. As is clear from the response curve in FIG. 4, it was shown that the biotinylated single chain antibody could be immobilized on streptavidin and still retain the function of binding an antigen. As shown in Table 1, the dissociation equilibrium constant Kd was calculated based on this curve, and it was found to be on the order of 1×10 ⁻⁷ to 10 ⁻⁸ M. From this result, it was revealed that the single-chain antibody prepared in Example 1 and immobilized by binding of biotin and streptavidin has a Kd value equivalent to that of the complete anti-Salmonella antibody IgG.

Comparative Example 1 Examination of immobilization efficiency by biotin binding position Addition of biotin by chemical binding to single chain antibody This method is described in the following biochemistry experimental course, immunobiochemistry research method (Japan Biochemical Society, Tokyo Kagaku Dojin (1986). )) The method described in the antibody labeling method was used.
The reaction solution synthesized by the method described in Example 1 without adding biotin ligase and biotin during the translation reaction was centrifuged at 15,000 rpm for 10 minutes to obtain a supernatant. After diluting 25 μl of the soluble fraction of the supernatant with the same amount of a 1 M sodium hydrogen carbonate solution, the buffer solution was exchanged with a G-25 Sephadex column, and 1 μl of NHS-biotin (N-hydroxysuccimide-biotin, 50 mg/ml) DMSO) was added. After reacting this at 4° C. overnight, the binding property with an antigen was analyzed as shown below.
Analysis of Binding Activity with Antigen 30 μl of the reaction solution prepared in (1) above was subjected to gel filtration of excess biotin with G-25 spin column equilibrated with 10 mM PBST (pH 8.0) and 0.6 mM CaCl ₂ . 40 μl of the protein solution was washed with 25 μl of a wheat germ extract containing 0.6 mM CaCl ₂ in advance and 96 μl together with 10 μl of the immobilized Salmonella sugar chain antigen (Sal-Mag) prepared in Example 2(1) above. It was added to the microplate. After gently mixing for 15 minutes, the plate was washed 4 times with 40 μl of 0.15 M NaCl/10 mM PBST (pH 8.0), and finally eluted with the same amount of 0.1 M glycine-HCl (pH 2.4) 4 times. It was The result is shown in FIG. If the antibody retains its activity, it should be eluted by the latter half of the acidic buffer, but as is clear from the figure, no protein is found in fraction numbers 10 to 13, and the first pass-through Most were present in the minute. This indicates that the biotinylated single chain antibody produced by the above chemical method has lost the antigen binding activity.
Example 3 Production and Immobilization of Single-Chain Antibody Having Polyhistidine Peptide Inserted (1) Production of Single-Chain Antibody Containing Polyhistidine Peptide in Linker Part The scfv-pEU described in Example 1(1) was used as a template. PCR was carried out using a LA taq (manufactured by TAKARA) kit with the primers consisting of the nucleotide sequences of SEQ ID NOs: 8 and 9. The PCR reaction solution was prepared in 5 μl 10×LA buffer, 5 μl 25 mM magnesium chloride, 8 μl 2.5 mM dNTP, 1 μl each 20 μM primer, 0.1 ng template plasmid/50 μl, 94° C. 1 min×1 cycle, 94° C. 45 sec. /55° C. 1 minute/72° C. 1 minute 30 seconds×30 cycles, 72° C. 5 minutes. The amplified DNA fragment was blunt-ended with KOD T4 polymerase (manufactured by NEB) according to a conventional method, then phosphorylated with Polynucleotide kinase (manufactured by NEB), and then self-ligated by Ligation high (manufactured by Toyobo). Ligation was performed to prepare a circular plasmid (FIG. 1: hereinafter, this may be referred to as “scFV-pHIS-pEU”).
Using this plasmid as a template, transcription was performed by the method described in Example 1(3) to purify mRNA, and then DTT in the translation reaction solution was replaced with 200 μM mercaptoethanol to perform translation reaction. The reaction solution after 3 hours of the translation reaction was centrifuged at 15,000 rpm for 10 minutes to separate the solubilized components, and an excess amount of mercaptoethanol was added to 50 mM phosphate buffer (pH 7.0), 500 mM NaCl, 5% Glycerol( It was removed by a G-25 spin column equilibrated with a binding buffer).
20 μl of the eluate of the spin column was diluted with the same amount of binding buffer, and then 80 μl was added to 200 μl (50% resin) of a nickel column (Metal affinity resin: manufactured by TALON) which was washed 6 times with 150 μl of binding buffer in advance. Incubated for 1 hour. This column was washed 4 times with 150 μl of 50 mM phosphate buffer (pH 7.0), 500 mM NaCl, 5% Glycerol, 6 mM Immidazole (washing buffer) (w1 to w4 in the figure), and further 150 μl of 50 mM phosphate buffer. Elution was performed 5 times (e1 to e5 in the figure) with (pH 7.0), 500 mM NaCl, and 150 mM Immidazole (elution buffer). The amount of single chain antibody contained in each fraction was measured by the 14C dpm value. The result is shown in FIG. In the figure, C indicates the 14C dpm value in the total amount of the protein-containing solution before being applied to the column. The horizontal axis of the graph indicates the fraction number, w1 to w4 indicate the 14C dpm peaks in the fraction eluted by the washing buffer, and e1 to e5 indicate the 14C dpm in the fraction eluted by the elution buffer. ET indicates the sum of 14C dpm values in e1 to e5.
Fraction numbers e1 to e5 indicate the presence of single chain antibodies containing polyhistidine peptides that specifically bind to nickel. As is clear from the figure, it was found that about 50% of the total synthetic amount of the single chain antibody can be purified by the nickel column. It was also confirmed by the method described in Example 2 that the purified single chain antibody retains the antigen binding activity. This result indicates that the single-chain antibody in which the polyhistidine peptide is incorporated in the linker portion can be immobilized on the nickel solid phase while maintaining its activity.

    According to the present invention, a single-chain antibody or a labeled single-chain antibody, which retains a specific binding activity with an antigen, is provided. The single-chain antibody can be bound to a solid phase via a labeling substance, and an antibody chip or the like can be produced. Such a single-chain antibody can be synthesized with a cell-free protein translation system in which the intramolecular disulfide bond is retained, so that it is more specific for the antigen than that synthesized in a living cell such as Escherichia coli. Those with high cohesiveness are provided.

[Sequence list]

Claims (27)

A single-chain antibody characterized by having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker, or a labeled single-chain antibody characterized by carrying a labeling substance on the linker portion of the single-chain antibody .. A single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker, or a labeled single-chain antibody having a labeling substance carried on the linker portion of the single-chain antibody, wherein the heavy chain and the light chain of the antibody Is a variable region, or a labeled single-chain antibody. A labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are crosslinked via a linker, and carrying a labeling substance on the linker part, wherein the labeling substance is an antibody in the presence of a specific enzyme. A labeled single-chain antibody, which is a substance capable of binding to the polypeptide of the linker moiety of. In a labeled single-chain antibody having a structure in which a heavy chain and a light chain that are variable regions of an antibody are cross-linked via a linker, and a labeling substance is carried on the linker portion, the labeling substance is a specific enzyme. A labeled single-chain antibody, which is a substance capable of binding to a polypeptide of a linker portion of an antibody in the presence thereof. In a labeled single chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker, and carrying a labeling substance on the linker part, the labeling substance is a part of the linker part of the antibody. A labeled single-chain antibody characterized by being incorporated. In a labeled single-chain antibody having a structure in which a heavy chain and a light chain, which are variable regions of an antibody, are cross-linked via a linker, and the linker moiety carries a labeling substance, the labeling substance is a linker part of the antibody. A labeled single chain antibody characterized in that it is incorporated as a part of Labeling having a structure in which the heavy chain and the light chain of an antibody are crosslinked via a linker, and carrying a labeling substance capable of binding to the polypeptide of the linker part of the antibody in the linker part in the presence of a specific enzyme A single-chain antibody, wherein the labeling substance is biotin and the enzyme is biotin ligase. The variable region of the antibody has a structure in which heavy and light chains are cross-linked via a linker, and a labeling substance capable of binding to the polypeptide of the linker part of the antibody in the presence of a specific enzyme is provided in the linker part. The labeled single-chain antibody carried, wherein the labeling substance is biotin and the enzyme is biotin ligase. The single chain antibody or the labeled single chain antibody according to any one of claims 1 to 8, which has a Kd value equivalent to that of a natural antibody and is produced by a cell-free protein translation system using wheat germ. A DNA characterized in that DNAs encoding heavy and light chains of an antibody having a binding property to a specific antigen are linked via a DNA encoding a linker. In the DNA in which the DNAs encoding the heavy chain and the light chain of the antibody having the ability to bind to a specific antigen are linked via the DNA encoding the linker, the heavy chain and the light chain of the antibody are variable regions DNA characterized by: In the DNA in which the DNAs encoding the heavy chain and the light chain of the antibody having the ability to bind to a specific antigen are linked via the DNA encoding the linker, the DNA encoding the linker is a DNA of a specific enzyme after translation. A DNA comprising a base sequence capable of binding a labeling substance in the presence thereof. In a DNA in which heavy chain and light chain that are variable regions of an antibody capable of binding to a specific antigen are linked via a DNA encoding a linker, the DNA encoding the linker is post-translationally translated. A DNA comprising a base sequence capable of binding a labeling substance in the presence of a specific enzyme. DNAs encoding heavy and light chains of an antibody capable of binding to a specific antigen are linked via a DNA encoding a linker containing a base sequence capable of binding a labeling substance after translation in the presence of a specific enzyme. DNA which is characterized in that the base sequence capable of binding the labeling substance encodes an amino acid sequence recognized by biotin ligase. DNA encoding a heavy chain and a light chain, which are variable regions of an antibody capable of binding to a specific antigen, and a linker containing a base sequence capable of binding a labeling substance in the presence of a specific enzyme after translation. A DNA characterized in that, in the DNA linked via, the base sequence capable of binding the labeling substance encodes an amino acid sequence recognized by biotin ligase. A method for producing a labeled single-chain antibody, which comprises transcribing and translating the DNA according to any one of claims 10 to 15 using a protein synthesis system in the presence of a labeling substance and a specific enzyme. A method for producing a single-chain antibody or a labeled single-chain antibody, which comprises transcribing and translating the DNA according to claim 10 or 11 using a protein synthesis system. The protein synthesis system is a wheat germ-derived cell-free protein translation system, and the concentration of the reducing agent in the translation reaction solution maintains the disulfide bond of the single-chain antibody to be produced and enables cell-free protein synthesis. The method for producing a single chain antibody or a labeled single chain antibody according to claim 16 or 17, wherein the concentration is the concentration. The method for producing a single chain antibody or a labeled single chain antibody according to claim 18, which is further carried out in the presence of an enzyme that catalyzes a disulfide bond exchange reaction. A single-chain antibody or a labeled antibody having a Kd value equivalent to that of a natural antibody produced by the method for producing a single-chain antibody or a labeled single-chain antibody according to claim 19 using a cell-free protein translation system derived from wheat germ. Single chain antibody. A solid-phase immobilized single-chain antibody, characterized in that the antibody described in any one of the following is brought into contact with a substrate partitioned into a plurality of regions having a substance that specifically binds to a labeling substance of the antibody on the surface. Production method.
1) A labeled single-chain antibody, which has a structure in which a heavy chain and a light chain of an antibody are crosslinked via a linker, and carries a labeling substance on the linker portion.
2) A labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker and carrying a labeling substance on the linker part, wherein the heavy chain and the light chain of the antibody are variable regions. A labeled single-chain antibody characterized in that
3) A labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker and carrying a labeling substance on the linker part, wherein the labeling substance is present in the presence of a specific enzyme. A labeled single-chain antibody, which is a substance capable of binding to the polypeptide of the linker portion of the antibody.
4) In a labeled single-chain antibody having a structure in which a heavy chain and a light chain, which are variable regions of an antibody, are cross-linked via a linker, and the linker moiety carries a labeling substance, the labeling substance is A labeled single-chain antibody, which is a substance capable of binding to a polypeptide of a linker portion of an antibody in the presence of an enzyme.
5) In a labeled single-chain antibody having a structure in which a heavy chain and a light chain of an antibody are cross-linked via a linker and carrying a labeling substance on the linker part, the labeling substance is one of the linker parts of the antibody. A labeled single chain antibody characterized by being incorporated as a part.
6) In a labeled single-chain antibody having a structure in which a heavy chain and a light chain, which are variable regions of an antibody, are cross-linked via a linker, and a linker is loaded with a labeling substance, the labeling substance is A labeled single-chain antibody characterized by being incorporated as part of a linker moiety.
7) The heavy chain and the light chain of the antibody have a structure in which they are crosslinked via a linker, and the linker part carries a labeling substance capable of binding to the polypeptide of the linker part of the antibody in the presence of a specific enzyme. A labeled single-chain antibody, wherein the labeling substance is biotin and the enzyme is biotin ligase.
8) Labeling having a structure in which heavy and light chains that are variable regions of an antibody are cross-linked via a linker, and capable of binding to the polypeptide of the linker part of the antibody in the presence of a specific enzyme in the linker part A labeled single-chain antibody carrying a substance, wherein the labeled substance is biotin and the enzyme is biotin ligase. 22. The method for producing a solid-phase immobilized single chain antibody according to claim 21, wherein two or more different types of solid-phase immobilized single chain antibodies are immobilized on a substrate divided into a plurality of regions. For producing a modified single chain antibody. 23. The production method according to claim 21 or 22, wherein the labeling substance is biotin, and the substance that specifically binds to the labeling substance is streptavidin. A solid-phase immobilized single-chain antibody prepared by the method according to any one of claims 21 to 23. A method for analyzing an antigen-antibody reaction, which comprises contacting a test substance with the immobilized single chain antibody according to claim 24 and analyzing the binding property to the immobilized single chain antibody. A method for analyzing an antigen-antibody reaction, which comprises the following steps.
(1) a step of preparing a labeled single-chain antibody under conditions in which the disulfide bond of the single-chain antibody is retained, including the following elements (1) or (2):
(1) A DNA encoding a linker containing a nucleotide sequence capable of binding a labeling substance in the presence of a specific enzyme after translation, which is a DNA encoding a heavy chain and a light chain of an antibody capable of binding to a specific antigen. A step of producing a labeled single-chain antibody by transcribing the DNA ligated via the transcription-translation using a wheat cell-free protein synthesis system in the presence of a specific enzyme,
(2) A DNA containing a heavy chain and a light chain, which are variable regions of an antibody capable of binding to a specific antigen, has a linker containing a base sequence capable of binding a labeling substance in the presence of a specific enzyme after translation. A step of producing a labeled single-chain antibody by transcribing and translating the DNA linked via the encoding DNA using a wheat cell-free protein synthesis system in the presence of a specific enzyme,
(2) Prepare a substance (adapter substance) that specifically binds to the labeled substance of the labeled single-chain antibody when the labeled substance of the labeled single-chain antibody is a solid-phase substance, including the following elements Process,
(1) Immobilizing a substance (adapter substance) that specifically binds to the labeling substance of the labeled single-chain antibody on a substrate divided into a plurality of regions,
(2) A step of removing a substance (adapter substance) that specifically binds to the labeling substance of the labeled single-chain antibody that is not fixed to the substrate of (1) above,
(3) A step of appropriately removing non-specific adsorption on the substrate before and after the step (1) or (2) above,
(3) a step of preparing a solid-phased labeled single-chain antibody in the case where the labeled substance of the labeled single-chain antibody is a solid-phased substance, including the following elements:
(1) A substance that specifically binds the labeled substance of the labeled single-chain antibody prepared in (1) or (2) above to the labeled substance of the labeled single-chain antibody of (2) (adapter substance) ) A step of adding and contacting a necessary amount with a substrate divided into a plurality of areas having a surface,
(2) A step of removing the labeled single-chain antibody that is not fixed to the substance (adapter substance) that specifically binds to the labeled single-chain antibody on the substrate of (1) above,
(3) A step of removing non-specific adsorption on the substrate as appropriate, following the step of (2) in the first half,
(4) a step of preparing a labeled single-chain antibody in the case where the labeled substance is a signal substance, including the following elements:
(1) A step of appropriately removing non-specific adsorption on the substrate divided into a plurality of regions,
(2) A step of adding a necessary amount of the labeled substance of the labeled single-chain antibody prepared in (1) or (2) above to a substrate,
(5) A step of adding a required amount of a test substance to each substrate described in (3) or (4) above, and analyzing the binding property between the labeled single-chain antibody and the test substance,
(6) A step of qualitatively or quantitatively determining the interaction between the labeled single chain antibody and the test substance based on the binding result of (5). A reagent kit for measuring an antigen-antibody reaction, which comprises a reagent used in the analysis method according to claim 25 or 26.

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